Probiotic Enriched and Low-Organic Acid Food Products

ABSTRACT

This invention relates to a fruit-based food product such as beverages or fruit purees, comprising a concentration of live and stable probiotics, which is preferably greater than 10 8  CFU/ml, a high fruit content, which is preferably greater than 50%, and a low organic acid content, and wherein the production of off-tastes is reduced or diminished in relation to the initial fruit matrix, as well as method for preparing a food product such as this.

This invention relates to a fruit-based food product such as beverages or fruit purees, comprising a concentration of live and stable probiotics, which is preferably greater than 10⁸ CFU/ml, a high fruit content, which is preferably greater than 50% and the organic acid content of which is reduced by 10 to 100%, preferably by 30 to 70%, and more preferably yet by 60%, in relation to the initial organic acid content of the fruit matrix, as well as a method for preparing a food product such as this.

The ingestion of live microorganisms called probiotics, some of which are bacteria, and in particular those which belong to the genus Lactobacillus, are particularly beneficial to one's health. As a matter of fact, they have been the subject matter of numerous studies demonstrating preventive clinical effects in various fields (e.g., in the fields of allergic manifestations, infectious diarrhoea and inflammatory diseases) and on certain physiological functions (e.g., the digestion of lactose, bowel transit and immunity). These probiotics are, in particular, capable of promoting proper functioning of intestinal flora, which are likely to affect the overall population. As a matter of fact, among other things, these bacteria produce other bacteriocins and lactic acid, which indirectly increase the digestibility of food products, promote intestinal peristalsis, and accelerate the evacuation of stools. Furthermore, these bacteria produce certain B-complex vitamins, and in general promote the absorption of vitamins and minerals, reduce blood cholesterol, strengthen the immune system and coat the mucous membranes of the intestines so as to protect against the invasion and activities of harmful microorganisms.

For this reason, the agroprocessing industries have been attempting for several years to incorporate such bacteria into their products.

Such products with added bacteria are traditionally dairy products, however, there is an advantage for the agroprocessing industry to develop other food products, particularly fruit-based ones.

Other fruit-based food products with added bacteria of the Lactobacillus type are already known in the prior art, e.g., in the international patent application WO 00/70972, and the European patent application EP 0113055.

However, it has been possible to observe bacterial growth in food products to which lactobacilli have been added, which, during storage of the products, results in the qualities thereof being altered by the production of gases and off-tastes, thereby making them unsuitable for consumption.

Numerous microorganisms are capable of decarboxylating substituted cinnamic acids such as trans-4-hydroxy-methoxy-cinnamic acid (ferulic acid) and trans-4-hydroxy-cinnamic acid (p-coumaric acid), in order to form the two following volatile compounds, respectively:

3-methoxy-4-hydroxystyrene (4-vinyl guaiacol) and 4-hydroxystyrene (4-vinyl phenol). These molecules are responsible for off-tastes of the “phenol, smoky, glove-like and medicinal” type. P-coumaric acid and decarboxylated ferulic acid activity was detected in bacteria of the Lactobacillus type. In particular, the lactobacilli known for these activities are as follows: L. brevis, L. crispatus, L. fermentum, L. plantarum, L. pentosus and L. paracasei (bibliog. Ref.: Van Beek, S and Priest F G —2000—“Decarboxylation of substituted cinnamic acids by lactic acid bacteria isolated during malt whisky fermentation—Applied and Environmental Microbiology, 66 (12): 5322-8). Strains of lactobacilli are thus capable of producing off-tastes from phenolic acids via biotransformation pathways.

At present, the solutions proposed in the prior art for solving this problem involving the production of gases and off-tastes consist of preserving the products at a temperature of between 4 and 8° C. and with a low fruit concentration (approximately 25%), for example, as in the international patent application WO 00/70972, filed by the PROBI company.

However, these solutions do not make it possible to propose products having a fruit concentration greater than 50%, and further comprising a significant concentration of live and stable bacteria of the Lactobacillus genus. A significant concentration is understood to mean a population greater than or equal to 10⁸ CFU/ml of product. Stable bacteria are understood to mean a bacterial population having reduced metabolic activity (production of gases and/or off-tastes, and acidification during limited and controlled storage conditions) when cold, i.e., at a temperature of between 4 and 10° C. The limited post-acidification is the result of a reduction in the concentration of organic acids present in the product, on the one hand, and, on the other hand, the low storage temperature of said product.

A fruit-based food product which is of the beverage or fruit puree type and which comprises stable, live probiotics, will have the advantage of providing the consumer with the benefits of fruits and probiotics.

The National Plan on Nutrition and Health advocates consumption of a minimum of five portions of fruits and vegetable per day. Observations conducted by numerous scientists show that consuming more fruits and vegetables makes it possible in particular, to reduce the cholesterol rate and lipid intake, and to limit the prevalence of obesity in children.

Several scientific studies suggest that probiotics can likewise play a leading role with regards to health. Each probiotic strain can offer specific health benefits. Among these benefits can be found: improved digestive system functioning and strengthened natural defenses. Some probiotics act by absorbing proteins and others produce vitamins. Some can likewise produce compounds that fight against the propagation of pathogenic bacteria and can therefore play a role in the intestinal ecosystem.

It would be desirable for the agroprocessing industry to be capable of preparing such food products, and that is the subject matter of this invention.

In order to increase the viability of bacteria, the European patent applications EP 0113055 and EP 0166238, filed by Kirin Beer, propose that the concentration of polyphenols in fruit juices, which are bacteriostatic components, be selectively reduced by placing the juices in contact with absorbing agents. In this case, the desired objective is to also promote fermentation of the bacteria and to not keep the initial population stable, as is the case according to this invention.

It is known in the prior art that fruits contain organic acids, and the inventors have observed that bacteria of the Lactobacillus genus metabolize these organic acids, and that this metabolization of organic acids can be responsible for the production of carbon dioxide and/or off-tastes in fruit-based products. The organic acid compositions of some fruits can also be known by consulting bibliographic sources, e.g., such as the acidity ranges per fruit in the “Code of Practice” of the A.I.J.N.

Numerous strains of the Leuconostoc, Streptococcus and Lactobacillus genera are capable of degrading malate, citrate, pyruvate, fumarate, tartrate and gluconate in order to produce gases. In comparison to the measurement of gas produced, the determination of diacetyl and acetoin contents is a more effective method for detecting pyruvate degradation (Hegazi, F. Z., Abo-Elnaga, I. G., 1980. “Degradation of organic acids by dairy lactic acid bacteria. Mikrobioligie der Landwirtshaft der Technologie und des Umweltschutzes, 135 (3), 212).

When degraded, organic acids such as malic acid or citric acid do not, as a matter of fact, present the consumer with these problematics involving the generation of unpleasant tastes, unless this assimilation is accompanied by an excessively high production of acetate, which also produces off-tastes. However, the assimilation of these organic acids by bacterial strains will in this case produce CO₂, which will inflate the product packaging. As a matter of fact, these organic acids are metabolized naturally by certain species of lactobacilli, in order to produce pyruvate (the principal compound of metabolic cycles such as carbonaceous metabolism) and CO₂; furthermore, pyruvate is itself subject to decarboxylation reactions, thus proportionally increasing the rates of CO₂ produced.

Some of these organic acids are phenolic compounds (coumaric acid, ferulic acid) and the degradation of these compounds by bacterial strains may produce off-tastes in the product.

Depending on the pH of the end product, the sensory profile of an acid is highly variable. Thus, lactic acid is more astringent at pH 3.5 in comparison to citric acid and malic acid (Hartwig, P., McDaniel, M. R., 1995. “Flavor characteristics of lactic, malic, citric and acetic acids at various pH levels.” Journal of Food Science, 60 (2), 384-388.).

FIG. 1 thus shows the mechanism of metabolism for malic acid (or malate), citric acid (or citrate) and pyruvate.

Commercial beverages exist, in particular the product ProViva® distributed by Skånemejerier, which contain a strain of the Lactobacillus genus at a maximum concentration equal to 10⁸ CFU/ml and at an acid pH (pH=3.8). However, the stability of the product is only guaranteed when stored at 4° C. and as the result of a low concentration of fruit juices (<25%). Furthermore, the ProViva® commercial products have targeted certain types of fruits which have low organic acid concentrations and which do not include major juices like orange juice, apple juice and exotic multi-fruit juices.

The inventors have shown that depletion of organic acids from the matrix of fruits on which the food product is based makes it possible to reduce or eliminate the production of carbon dioxide and/or off-tastes from the final food product, after packaging, while at the same time protecting the nutritional qualities of the product, regardless of the fruit, fruit concentration and organic acid concentration of the end product. Thus, the inventors propose to control and recommend a limiting organic acid concentration that must be adjusted to the fruit matrix target concentration in the formula and to the type of fruit in question.

One object of this invention, therefore, is a packaged fruit-based food product comprising live and stable probiotics and the organic acid content of which is reduced by 10 to 100%, preferably 30 to 70%, and more preferably still by 60% in relation to the initial organic acid content of the fruit matrix, and wherein the production of off-tastes is reduced or eliminated in comparison to the initial fruit matrix.

The term probiotic is meant to designate live microorganisms which, when incorporated in a sufficient quantity, have a positive effect on health, beyond traditional nutritional effects.

According to this invention, the term live probiotics is meant to designate probiotics for which the survival rate after 29 days in a food product according to this invention is greater than 60%, and advantageously greater than 80%.

The viability of probiotics is measured by counting techniques known to those skilled in the art, e.g., such as mass counting, surface counting, Malassez cells, direct counting, turbidity, nephelometry, electronic counting, flow cytometry, fluorescence, impedance measurement and image analysis.

According to this invention the term stable probiotics is meant to designate probiotics having an absence of activity for at least 30 days at 10° C. The absence of activity results in:

-   -   An absence of detection of gas production (e.g., CO₂) during         packaged storage,     -   A constant organoleptic quality without any alteration of the         initial qualities of the fruit matrix and without any production         of off-tastes,     -   An absence of significant post-acidification (decrease in pH<0.5         unit),     -   A non-proliferation of the probiotics and retention of the         initial population (±50%).

In particular, these probiotics can be bacteria.

According to this invention, the term bacteria is understood to preferably designate lactic bacteria, of the genera Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., Leuconostoc spp. and, in particular, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus aciophilus, Bifidobacterium animalis, Bifidobacterium breve, Streptococcus thermophilus and Lactococcus lactis.

More particularly, the preferred bacteria according to this invention are bacteria having the capacity to degrade organic acids into CO₂ and/or into compounds producing off-tastes.

More particularly, the preferred bacterial strains according to this invention are of the genus Lactobacillus, preferably Lactobacillus plantarum and Lactobacillus casei, and more preferably yet the Lactobacillus plantarum strains deposited at the Deutsche Sammlung von Mikrooganismen von Zelkuturen GmbH on 16 Mar. 1995, under the number DSM 9843, or strains of Lactobacillus plantarum deposited at the Collection Nationale des Cultures de Microorganismes on 4 Apr. 2002, under the number CNCM I-2845.

The Lactobacillus plantarum strain deposited at the Deutsche Sammlung von Mikrooganismen von Zelkuturen GmbH on 16 Mar. 1995, under the number DSM 9843 is marketed by the PROBI company, under the name Lactobacillus plantarum 299v®. This strain has numerous advantages for use as a probiotic in a fruit-based food product:

-   -   It meets the probiotic criteria established by the scientific         community.     -   It is patented, characterised (RAPD, ribotyping), and its         classification is confirmed;     -   It is GRAS (Generally Recognized as Safe);     -   It is already present at a rate of 10⁸ CFU/ml in the ProViva®         product distributed by Skånemejerier and has been consumed since         1994;     -   It has a very good survival rate at an acid pH lower than 4;     -   It is amylase-negative and therefore does not degrade the         texture of the end product.

However, this strain also has several disadvantages:

-   -   It has a strong post-acidification potential.     -   It results in significant organoleptic defects associated with         acetic acid synthesis.     -   It degrades citric acid (e.g., lemon juice, orange juice) or         malic acid (e.g., apple or pear juice), thus producing carbon         dioxide gas, hence possible swelling problems, particularly if         the cold chain is broken (i.e., going above the temperature of         8° C.).

Thus, this strain has numerous positive points, but it cannot be used as is in fruit-based food products, without depleting the organic acids of the fruit matrix on which the food product is based.

The same holds true for the strain of Lactobacillus plantarum deposited at the Collection Nationale des Cultures de Microorganismes on 4 Apr. 2002, under the number CNCM I-2845.

According to the invention, the term fruit matrix is understood to designate a fruit juice, a reconstituted fruit juice concentrate, or a fruit puree, without any probiotics, not depleted of organic acids, but optionally comprising other substances, e.g., such as sugar, water, flavourings, food colouring agents, sweetening agents, antioxidants, milk, preservatives, acidifiers, texturing agents, animal proteins (milk proteins, whey proteins . . . ) or vegetable proteins (soy, rice . . . ) or vegetable extract proteins (soy, rice . . . ).

The term off-tastes is understood to mean a taste that is abnormal for the food product. An off-taste is unpleasant to the consumer, and therefore not desirable. Thus, to illustrate, for the food product according to the invention, it is possible to cite the “earthy hay-like” off-taste resulting from the fermentation and oxidation of the product, the “vinegar-like” off-taste resulting from the organic acid ferment present in the product, and the “rancid” off-taste resulting from the presence of volatile fatty acids.

So-called “positive” notes can also be detected in the product, e.g., such as “orange” or “fruity” notes. Since these tastes are not unpleasant to the consumer, they are not included in the “off-tastes” according to this invention.

The concentration of molecules responsible for “off-tastes” is measured by solid phase microextraction (SPME) associated with gas chromatography (GC) coupled with a mass spectrometer (SM). This method was developed specifically and has enhanced sensitivity while at the same time having good reproducibility and good repeatability. SPME enables a specific concentration of the volatile target molecules for improved quantification and improved identification. CG enables separation of the volatile molecules based on the polarity and molar mass thereof, and thus obtainment of the peaks corresponding to each molecule. The concentration of each molecule is expressed in peak areas, i.e., in absorbance units (UA) proportional to their concentration in the sample. Finally, on the one hand, mass spectrometry enables positive identification of each molecule via the fragmentation thereof into characteristic ions, and, on the other hand, a second quantification of the volatile molecules wherein, this time, the concentration is expressed in mass units.

The term flavourings is meant to understand ingredients intended to give a flavour (i.e., a taste and/or an odour) to a foodstuff.

Flavourings are used for two primary objectives:

-   -   either they enhance the natural flavour of the food product, or         partially restore it if it is too weak (products having lost a         portion of their taste during the manufacturing process),     -   or they replace an ingredient while introducing an aromatic note         to the end product (e.g., strawberry flavoured yoghurt).

According to this invention, the preferred flavourings are: apple, orange, red berries, strawberry, peach, apricot, plum raspberry, blackberry, currant, lemon, citrus fruits, grapefruit, banana, pineapple, kiwi, pear, cherry, coconut, passion fruit, mango, fig, rhubarb, melon, multi-fruits, exotic fruits, vanilla, chocolate, coffee and cappuccino.

The term (food) colouring agents is understood to mean substances capable of restoring or giving colouring to the food product.

According to this invention, the preferred colourings are: beta-carotene and carmine.

The term sweetening agent is understood to mean substances capable of mimicking the sweetening power of sugar without necessarily introducing the calories of sugar.

According to this invention, the preferred sweetening agents are: aspartame, Acesulfame-K, saccharine, sucralose and cyclamate.

The term antioxidants is understood to mean substances capable of preventing or reducing the oxidation phenomena which, among other things, cause the rancidification of fats or the browning of cut fruits and vegetables.

According to this invention, the preferred antioxidants are: vitamin C, vitamin E and rosemary extract.

The term milk is understood to mean milk of animal origin (e.g., cow, goat and ewe) or juices of vegetable origin (e.g., juice extracted from soybeans, tofu, rice, oats, quinoa, chestnuts, almonds or hazelnuts).

The term preservatives is understood to mean substances intended to assist in preservation by preventing the presence and development of undesirable microorganisms (for example: moulds or bacteria responsible for food poisoning) in the final food product.

According to the invention, the preferred preservatives are sorbic acid and sulphur dioxide.

The term texturing agents is understood to mean substances which make it possible to improve the presentation or behaviour of the final food product. The texturing agents can be emulsifiers, stabilizers, thickening agents or gel-forming substances. They can be used in the food product according to the invention alone or in combination.

According to this invention, the preferred texturing agents are: pectin, used as a gel-forming substance, carob seed, carrageenans, alginates, guar gum, xanthan gum, starch and mono- and diglycerides of edible fatty acids.

The term acidifiers is preferably understood to mean lactic acid and/or citric acid and/or phosphoric acid.

The term water is optionally understood to mean osmosis-purified water. Osmosis-purified water makes it possible to limit the amount of minerals present in the end product, since minerals can likewise be responsible for off-tastes.

Potassium, chlorine, magnesium and calcium are in fact rather bitter in various forms (KCl, NH₄CL, CaCl₂, Calcium acetate LiCl, MgSO₄ . . . ) whereas sodium, lithium and sulphate are rather salty and/or acidic depending on the form thereof (salty form: NaCl₂, Na₂SO₄, Na tartrate; acidic form: Na₂NO₃, Li acetate; salty and acidic form: Na acetate, Na ascorbate, Na citrate). Besides these direct effects on the sensory qualities of the products, these compounds can also have a “salting out” effect on the volatile molecules responsible for “smoky, phenolic . . . ” off-tastes, by promoting the transition thereof to the gaseous phase above the product, thereby increasing the intensity of the off-tastes perceived.

According to this invention, the term organic acids is understood to designate, in particular, malic acid, citric acid, tartaric acid, pyruvic acid, fumaric acid or gluconic acid.

The organic acids the content of which is reduced or eliminated in comparison with the initial fruit matrix are preferably the malic and/or citric acids.

The initial organic acid content of the fruit matrix can be known from bibliographic sources. In the case where the fruit matrix is a fruit juice or a reconstituted fruit juice concentrate, the bibliographic references concern the organic acid concentration of the fruit juices. Such sources, for example, are the tables extracted from the AIJN “Code of Practice on Absolute Quality Requirements for Juices,” such as the one presented herein below:

TABLE 1 extract from the AIJN “Code of Practice on Absolute Quality Requirements for Juices,” showing the organic acid concentration of fruit juices. Citric acid (g/L) L-malic acid (g/L) Orange 6.7-17  0.8-3   Grapefruit  8-20 0.2-1.2 Apple  50-200 Min 3 Grape 0.5 max 2.5-7   Pineapple  3-11 1.0-4.0 Lemon 45-63 1.0-7.5 Passion fruit 25-50 1.3-5.0 Pear 4 max 0.8-5   Apricot 1.5-16   5-20 Black currant 26-42 1-4 Morello cherry 0.4 max 15.5-27   Raspberry  9-22 0.2-1.2 Strawberry  5-11 0.6-5   Peach 1.5-5   2-6 Mandarin orange  6-22 0.5-3  

In the case where the fruit matrix is not a fruit juice or a reconstituted fruit juice concentrate, the bibliographic sources concern the organic acid concentration of the fruits. Such sources, for example, are “La composition des aliments: tableaux des valeurs nutritives.” 2000 (6ème Edition); Souci S. W.; Fachmann W.; Kraut H.; Scherz H., a sample table of which is reproduced herein below:

TABLE 2 organic acid concentration of fruits Citric acid (mg/100 g) Malic acid (mg/100 g) Fruit Mean Min Max Mean Min max Pear 140 80 200 170 100 240 Strawberry 748 670 940 303 90 340 Peach 240 160 320 330 280 370 Pineapple 630 580 670 94 87 100 Grape 23 327 220 650 Apple 29 9 30 426 270 790 Apricot 400 140 700 1000 700 1300 Orange 1042 600 1880 89 40 190 Banana 201 80 390 360 240 500 Mango 264 200 327 74 Morello cherry 4.7 1800 Cherry (sweet) 13 10 15 940 730 1110 Plum 34 23 55 1220 820 1990 Prune 158 5690 Blackberry 18 15 21 900 860 950 Blueberry 523 850 Raspberry 1720 1060 2480 400 0 800 Grapefruit 1296 1000 1460 180 50 310 Guava 537 532 541 325 182 469 Kiwi 995 980 1010 500 470 530 Passion fruit 3250 650 Papaya 54 29 100 29 27 31 Lemon 4683 3500 7200 200 Quince 930 Rosehip 3100 Lychee 16 239 Pomegranate 500 100 Melon 75 0 150 0 50

In both cases, the value from the table that is used to determine the initial organic acid content of the fruit juices or fruits is the minimum value.

Furthermore, the initial organic acid content of the fruit matrix can be defined by any appropriate quantification method.

Such methods are, for example:

-   -   Measurement of the titratable acidity, which quantifies         concentration of acids present in the fruit matrix. This         consists in neutralizing a sample of the fruit matrix with a         0.1N sodium hydroxide solution, the amount of sodium hydroxide         required in order to reach a pH level of 8, thereby making it         possible to deduct the total acidity value.     -   Chromatographic assay using HPAEC (High Performance Anion         Exchange Chromatography) (Dionex method: 164-166 avenue Joseph         Kessel 78960 Voisins Le Bretonneux France) coupled with         conductimetric detection.     -   The malic and citric acids can be assayed via enzymatic methods;         the reference methods are advocated by the International         Federation of Fruit Juice Producers (IFU) (these references have         been established since 1985): IFU 21 for malic acid and IFU 22         for citric acid; these are spectrophotometric methods which         involve enzyme reactions.

The IFU 21 method operates on the following principle: the citric acid (citrate) initially present in the fruit matrix is converted into oxaloacetate and acetate in the reaction catalyzed by the citrate lyase enzyme CL (1).

In the presence of the L-malate dehydrogenase and L-lactate dehydrogenase enzymes, the oxaloacetate and pyruvate produced via decarboxylation, are reduced to L-malate and L-lactate, respectively, by Nictinamide Adenine Dinucleotide (NADH) (2) (3)

The amount of NADH oxidized in the reactions (2) and (3) is stoichiometric with the amount of citrate. NADH is determined by measuring the light absorbency thereof at 334, 340 or 365 nm. This measurement made it possible to determine the amount of citric acid initially present in the fruit matrix.

The IFU 22 method operates on the following principle: the L-lactic acid (L-lactate) initially present in the fruit matrix is oxidized into pyruvate with Nicotinamide Adenine Dinucleotide (NAD) in the presence of L-lactate dehydrogenase (L-LDH) (1).

The equilibrium for this reaction rests on the L-lactate side. By trapping the pyruvate in a subsequent reaction catalyzed by the glutamic-pyruric transaminase (GPT) enzyme in the presence of L-glutamate, the equilibrium can be shifted in favour of the pyruvate and NADH (2).

The amount of NADH formed in the previous reaction is stoichiometric with the amount of L-lactic acid. The increase in NADH is determined by measuring the light absorbency thereof at 334, 340 or 365 nm. This measurement makes it possible to determine the amount of L-lactic acid initially present in the fruit matrix.

According to a first aspect of the invention, the food product can be a beverage, preferably containing fruit juices or reconstituted concentrate-based fruit juices.

According to this invention, the following can be cited as fruit juices: orange juice, and in particular 10-12° Brix NFC (Not from Concentrate) and, as a reconstituted orange juice concentrate, FCOJ (Frozen Concentrate Orange Juice) at 66° Brix, and the other fruit juice concentrates at between 10 and 70° Brix.

According to this invention, the food product contains between 20 and 99.99% fruit juices, and preferably between 50 and 99.99% fruit juices.

According to a second aspect of this invention, the food product can be a fruit-based puree preferably comprising between 50 and 99.99% fruit purees and more preferably yet between 90 and 99.99% fruit purees.

According to this invention, the probiotics are at a concentration of between 5.105 and 1.109 CFU/ml, and preferably at a concentration greater than or equal to 10⁸ CFU/ml. The concentration is most preferably 4.107 CFU/ml.

According to the invention, the food product has a pH of between 3 and 4.

According to this invention, the food product keeps, and can therefore be consumed, for at least 30 days at a maximum temperature of 10° C., without requiring the addition of bacteriostatic agents.

According to this invention, the food product is one fruit-based.

According to this invention, the food product is based on several fruits.

According to this invention, the fruit or fruits have a high organic acid content.

According to this invention, the fruits are: orange, lemon, grape, pineapple, apple, pear, peach and/or red berries.

According to this invention, the food product preferably contains milk and/or vegetable juice.

The vegetable juice is preferably a juice made from soybean (juice extracted from soybeans and/or tofu).

According to this invention, the organic acids preferably eliminated from the fruit matrix are malic acid, citric acid, tartaric acid, pyruvic acid, fumaric acid, gluconic acid, p. coumaric and/or caffeic acid.

A second object of this invention is a method of preparing a food product according to this invention, characterised in that it includes the following steps:

-   -   a) Depletion of organic acids from the fruit-based matrix,     -   b) Addition of probiotics to the matrix obtained after step a),     -   c) Packaging of the product obtained after step b).

According to this invention, step a) for depleting organic acids from a fruit-based matrix is carried out by selecting a fruit matrix having low natural acidity.

According to this invention, the term fruit matrix having low natural acidity is understood to designate a fruit matrix from which a naturally low-acid juice is obtained, the acidity of which is between the low value indicated in the AIJN “Code of Practice,” (The Association of the Industry of Juice and Nectars from Fruits and Vegetables of the European Union) recognized by all professionals in the field of fruit juices, and −50% of this value.

Natural acidities depend not only on the fruit, but also on the variety thereof, the climate and the time of harvesting. Consequently, ranges of acidity per fruit are defined and the values are shown in the table below: (source: AIJN):

TABLE 3 ranges of acidity for fruit, according to the AIJN Titratable acidity (expressed in g of anhydrous citric acid per litre of Fruits juice, measured at pH 8.1 Orange 5-15 Grapefruit 7.7-18.5 Apple 2.2-7.5  Grape 4-11 Pineapple 3.2-11.5 Lemon 44.8-62   Passion fruit 25.6-50   Pear 1.4-7 g/kg Apricot 6.4-19.2 g/kg   Currant 26.7-40.1  Sour cherry 12.8-22.6  Raspberry 12.2-20   Strawberry  5.1-11.5 Peach 3.2-8 g/kg Banana  2-3.8 Mandarin orange 5.8-19.2

As concerns the orange, the varieties found to have low natural acidity can have an acidity level of 3, for example, (i.e., 40% below the low value of the range).

According to this invention, the selection of a fruit matrix having a low natural acidity is carried out via varietal selection of the fruits and/or by controlling the ripening of the fruit.

The fruits will preferably be selected as soon as they have reached late maturity.

According to this invention, step a) for depleting organic acids from a fruit-based matrix is carried out via de-acidification of the fruit matrix.

According to this invention, de-acidification (reduction in titratable acidity) of the fruit matrix is carried out via electrodialysis of the fruit matrix, precipitation of the organic acids from the fruit matrix with calcium salts, malolactic fermentation, selective assimilation of the citric acid and/or passing the fruit matrix over an anion exchange resin.

Citric fermentation results in the production of diacetyl and acetoin via lactic bacteria.

De-acidification of the fruit matrix according to the invention is preferably carried out via electrodialysis and/or passage of the fruit matrix over an anion exchange resin.

As a matter of fact, an anion exchange resin is ideal for collecting compounds which have COOH acid radicals, since these radicals are easily separated into COO⁻ (anion) and H⁺ (cation), and said resin is therefore suitable for collecting organic acids.

The anion exchange resins used, for example, can consist of Dowex® 1 resins supplied by Dow Chemical, USA and Amberlite® IRA-402 resins supplied by Rohm and Haas Co., USA.

According to this invention, the probiotics are incorporated as part of a delayed differentiation process, i.e., at the end of the production line and immediately before or during the packaging step.

Furthermore, step b) and step c) according to this invention can be carried out simultaneously. In this hypothetical case, the method according to the invention only has two steps for preventing alterations of the final food product during storage thereof, without any proliferation of microorganisms.

In one preferred embodiment, a step for adding lactic acid is carried out between step b) and step c) of said method, or simultaneously therewith. The amount of lactic acid to be added will be easily determined by those skilled in the art, based on the desired strain of bacteria to be used.

FIGURE LEGEND

FIG. 1: Diagram of the metabolic assimilation of citrate and malate, and of the production of acetate in lactic bacteria.

EXAMPLES Example 1 Formation of Gases by the L. plantarum DSM 9843 and L. plantarum I-2845 Strains (Deposited at the CNCM on 4 Apr. 2002) in Relation to the Inoculated Fruit Juice Material and Methods:

I.1. Preparation of the Bacterial Suspensions and Inoculation of the Fruit Juices

A first 2-mL pre-culture is made with the DSM 9844 and I-2845 strains. This pre-culture serves to seed 100 mL of 1% neutral SRM (i.e., 10⁸-10⁹ CFU/mL). From this second pre-culture, 3×1,000 mL neutral SRM (i.e., 108-10⁹ CFU/mL) are seeded.

Centrifuging (Beckman JA-25, rotor JA-10) is carried out for each strain, as follows, using 500-mL bowls:

-   -   filling 6 bowls with 330 mL of culture,     -   centrifuging for 10 min, 12,000 g, 20° C.,     -   elimination of the supernatant and addition of 165 mL of         culture,     -   centrifuging for 10 min, 12,000 g, 20° C.,     -   elimination of the supernatant.

Each pellet obtained is then taken up again separately in the fruit juice being tested, and the suspension obtained is put back into the fruit juice brick carton, which is then closed back up again carefully.

I.2. Organic Acid Assays

The chosen technique consists in separating the organic acids via high performance anion exchange chromatography (HPAEC). Detection of the organic acids is carried out by suppressive conductimetric detection (SCD).

The chromatographic system used is of the DIONEX brand (DX600 type) comprising a suppressive conductimetric detection system. The thermostatically controlled conductimetric cell (DS3 type) is coupled to an external self-suppression system ASRS-ULTRA (4 mm). This electrolytic suppressor was used with a Milli-Q counter-current water recirculation means, at a flow rate of 4 mL/min (approximate pressure of 15 psi).

An AS11-HC-type (4 mm) anion exchange column is associated with an AG11-HC-type guard column. The elution flow rate is 1.5 mL/min.

II Results:

II.1. Bacterial Counts

Bacterial counts are carried out during storage of the products, so as to evaluate the survival of L. plantarum during storage of the fruit juices at 10° C.

TABLE 4 Bacterial counts of L. plantarum during storage of the fruit juices at 10° C. Time Strain (d) Orange Apple Grape DSM DO 1.8 · 10⁹ 1.7 · 10⁹ 9.5 · 10⁸ CFU/mL CFU/mL CFU/mL 9843 D5 5.0 · 10⁹ 5.8 · 10⁸ 4.1 · 10⁹ CFU/mL CFU/mL CFU/mL I2845 D0 1.1 · 10⁹ 9.8 · 10⁸ 6.0 · 10⁸ CFU/mL CFU/mL CFU/mL D5 4.5 · 10⁹ 1.6 · 10⁹ 3.9 · 10⁹ CFU/mL CFU/mL CFU/mL

II.2. Demonstration of the Consumption of Organic Acids During Storage.

The organic acid assays were carried out at 0 and 5 days, at the same time as the counts, and the results are summarized in Table 4.

TABLE 5 Metabolites produced and organic acids consumed when the fruit juice containing L. plantarum was stored at 10° C. Swelling Lactate Acetate Malate Citrate of the produced produced consumed consumed Batches bottle pH mmol mmol mmol mmol Apple Control + D0 − 3.43 0.00 0.00 0.00 0.00 juice DSM D5 ++ 3.38 27.93 4.44 6.72 0.21 9843 + I-2845 D5 ++ 3.39 40.86 4.38 21.20 0.19 Orange Control + D0 − 3.34 0.00 0.00 0.00 0.00 juice DSM D5 +++ 3.26 53.79 25.78 11.99 4.91 9843 + I-2845 D5 ++ 3.27 48.90 15.60 13.26 1.42 Grape Control + D0 − 3.22 0.00 0.00 0.00 0.00 juice DSM D5 ++ 3.23 40.79 10.38 23.50 2.09 9843 + I-2845 D5 +++ 3.24 44.59 8.16 33.87 1.43

According to the results presented in Table 5, it is quite apparent that malic acid is the substrate most heavily consumed by L. plantarum, regardless of the strain involved. This consumption is not only accompanied by production of lactate and acetate, and thus a noticeable decrease in the pH level (particularly in orange and apple juice) but also a production of gas which has a macroscopic effect on the packaging.

According to the metabolic pathways presented in FIG. 1, the absence of detection of formate production (no pyruvate formate-lyase action), the very low pentose content in the fruit juices treated, the following batch reconciliation of CO₂ (expressed in moles) can be proposed:

Total CO₂=malate consumed+citrate consumed+(total acetate produced−citrate-derived acetate).

Thus, by replacing the acetate produced from the citrate by the amount of consumed citrate:

Total CO₂=malate consumed+total acetate produced.

Conclusion:

Thus, malic acid and, to a lesser extent, citric acid contribute heavily to the production of gases when fruit juices containing a high dose (>1.10⁹ FCU/mL) of DSM 9843 or I-2845 L. plantarum bacteria.

Example 2 Dilution of Orange Juices in Order to Define the Maximum Concentrations of Organic Acids Compatible with L. plantarum in Relation to the Percentage of Juice in the Formula

We made dilutions with 5, 10, 20 and 30% orange juice, these dilutions corresponding to the de-acidification rates of 95, 90, 80 and 70%.

TABLE 6 % of juice in the % of de- Swelling product acidification pH observed? 30% 70% Natural pH Swelling at D + 3 30% 70% 3.7 Swelling at D + 5 20% 80% Natural pH Slight swelling at D + 14 20% 80% 3.7 Very slight swelling 10% 90% Natural pH No 10% 90% 3.7 No  5% 95% Natural pH No  5% 95% 3.7 No

In order for an orange juice-based beverage containing L. plantarum to be stable more than 30 days after the manufacture thereof, we determined that it was necessary for it to have the following characteristics:

TABLE 7 % of orange juice Acidity of the in the beverage orange juice Brix/acidity ratio 100 0.4-0.6 100-150 75  1.3-1.45 41-46 50 2.2-2.3 27-30

Comments:

-   -   the “Brix/acidity ratio” designates the ratio of the Brix value         of the juice to the number of grams of anhydrous citric acid per         100 grams of juice; (Brix/acid ratio).     -   the “Brix value” designates the sugar content determined by         refractometry, to which the acidity correction is added,         according to the method of the Association of Official         Analytical Chemistry of the United States, entitled “Solids         (Soluble) in Fruit Products,” published in Official Methods of         Analysis of the Association of Official Analytical Chemistry,         14^(th) Edition, 1984. (Brix content) DORS/88-8, art. 2;         DORS/95-548, art. 2; DORS/2000-184, art. 27; DORS/2003-6, art.         65 (F).

Example 3 Sensory Analysis of Various Products

Subsequent to the technical problems of using the DSM 9843 strain in plant environments (production of CO₂ via the metabolization of malic and/or citric acid, resulting in swelling of the UHT brick cartons; production of off-tastes due to the presence of organic acids and the metabolization of phenolic acids), the following technical solutions were tested:

1) De-acidification of the juices on ion-exchange resins,

2) Use of osmosis-purified water (to evaluate the impact of minerals on off-tastes),

3) Use of various types of acids for acidification: lactic acid, citric acid or phosphoric acid.

7 tests were conducted from all of these hypotheses:

TABLE 8 Type of orange Presence of Type of acid Tests juice (24%) milk (16%) for pH 3.8 SLC Standard juice Yes (L) Citric acid (C) DLC De-acidified Yes Citric acid juice (D) SLL Standard juice Yes Lactic acid (L) DLL De-acidified Yes Lactic acid juice DLL (osmosis- De-acidified Yes Lactic acid purified juice DL De-acidified No Lactic acid juice LL No juice Yes Lactic acid

Results:

TABLE 9 Type of Flavours Flavours at BALANCE MIX acid Test at Day 0 D 30 SHEET Standard + + SLC “Juice” “Juice” notes + ↓ juice milk citric notes +++ off-tastes +++ “Juice” acid off-tastes− (hay, soil, notes stable) ↑↑↑ off- tastes + + SLL “Juice” “Juice” notes + ↓ milk lactic notes +++ off-tastes +++ “Juice” acid off-tastes− (hay, soil, notes stable) ↑↑↑ off- tastes De- + + DLC “Juice” “Juice” notes − ↓ acidified milk citric notes +++ off-tastes +++ “Juice” juice acid off-tastes− (vinegar) notes ↑↑↑ off- tastes + + DLL “Juice” “Juice” notes − No milk lactic notes − off-tastes + “juice” acid off-tastes−− (rancid) notes ↑ off- tastes + + Osmosis − “Juice” “Juice” notes − No milk lactic purified notes − off-tastes − “Juice” acid DLL off-tastes−− (somewhat notes fruity juice) no off- tastes + + DL “Juice” “Juice” notes − ↓ milk lactic notes +++ off-tastes − “Juice” acid off- (somewhat notes tastes −−− fruity juice) no off- tastes Milk + LL “Juice” “Juice” notes −− No lactic notes −− off-tastes −−− “juice” acid off-tastes−− (very notes fruity/floral) no off- tastes Comment: the flavours are evaluated from − (very weak intensity for this flavour) to +++ (very strong intensity for this flavour). A downward arrow in the table (↓) indicated a reduction, and upward arrow (↑) an increase, and three upward arrows (↑↑↑) a significant increase in the positive and/or negative aromatic notes (“juice” notes) (off-tastes) for the juice.

Generally speaking, even though citric acid strengthens the “orange juice” notes in the products from D0 to D30, all of the products lose these organoleptic characteristics and are relative neutral as concerns the fruity/orange notes.

From a metabolic perspective, citric acid is indeed a precursor of off-tastes, because this acid is metabolized by Lactobacillus plantarum in order to form acetic acid (in the case of de-acidified juice) or ethyl phenol (in the case of standard juice). Consequently, the addition of citric acid should be as small as possible in order to prevent the formation of these molecules, while at the same time having a positive effect on the “juice” notes.

With regard to the type of juice, the de-acidification of juices is a method that makes it possible to prevent the formation of off-tastes (except in the presence of citric acid) and only rancid notes persist, primarily due to the presence of milk. Consequently, an adjustment in the de-acidified juice/milk ratio should be made.

Finally, the best results, in terms of an absence of off-tastes (hay, stable, soil, vinegar, rancid) and the presence of aromatic notes characteristic of orange juice, are obtained with de-acidified juiced mixed with osmosis-purified water and acidified with lactic acid.

Example 4 Fruit Juice+Milk Formula

TABLE 10 Orange fruit juice + milk product at a target pH of 3.8 Water Approximately 70% Sugar Approximately 7.5% De-acidified orange juice Approximately 4.5% concentrate Orange flavour 0.0054% Pectin 0.56% Beta-carotene 0.09% Milk Approximately 16.5% Lactic acid Final pH 3.8 Lactobacillus plantarum DSM Seeded at 0.1% 9843

Example 5 50% and 75% Orange Juice Formulas

TABLE 11 50% Orange fruit juice at a target pH of 3.6 Water 80.4 Sugar 3.5 De-acidified orange juiceconcentrate 11 Pulp 3.7 Pectin 0.2 Food colouring agent 0.02 Lactic acid 0.4 Ascorbic acid 0.03 Strain of L. plantarum DSM Seeded at 0.1% 9843

TABLE 12 75% Orange fruit juice at a target pH of 3.6 Water 80.2 Sugar 1.3 De-acidified orange juice 13.4 concentrate Pulp 3.9 Pectin 0.2 Food colouring agent 0.02 Lactic acid 0.13 Ascorbic acid 0.03 Strain of L. plantarum DSM Seeded at 0.1% 9843 FIGURES 

1. Packaged, fruit-based food product comprising live and stable probiotics the organic acid content of which is reduced by 10 to 100%, in relation to the initial organic acid content of the fruit matrix and wherein the production of off-tastes is reduced or eliminated in relation to the initial fruit matrix.
 2. Food product according to claim 1, characterised in that it is a beverage.
 3. Food product according to claim 2, characterised in that the fruit matrix is a fruit juice or a concentrate-based reconstituted fruit juice.
 4. Food product according to claim 1, characterised in that it contains between 20 and 99.99% fruit juices.
 5. Food product according to claim 1, characterised in that it contains between 50 and 99.99% fruit juices.
 6. Food product according to claim 1, characterised in that it is a fruit-based puree.
 7. Food product according to claim 1, characterised in that it contains between 50 and 99.99% fruit puree.
 8. Food product according to claim 7, characterised in that it contains between 90 and 99.99% fruit puree.
 9. Food product according to claim 1, characterised in that the probiotics are bacterial strains of the genus Lactobacillus.
 10. Food product according to claim 9, characterised in that the probiotics are Lactobacillus plantarum.
 11. Food product according to claim 10, characterised in that the probiotics are strains of Lactobacillus plantarum deposited at the Deutsche Sammlung von Mikrooganismen von Zelkuturen GmbH on 16 Mar. 1995, under the number DSM 9843, or strains of Lactobacillus plantarum deposited at the Collection Nationale des Cultures de Microorganismes on 4 Apr. 2002, under the number CNCM 1-2845.
 12. Food product according to claim 1, characterised in that the probiotics are at a concentration of between 5.105 and 1.108 CFU/ml.
 13. Food product according to claim 12, characterised in that the probiotics are at a concentration greater than or equal to 10⁸ CFU/ml.
 14. Food product according to claim 1, characterised in that the pH thereof is between 3 and
 4. 15. Food product according to claim 1, characterised in that it keeps for at least 30 days at a maximum temperature of 10° C.
 16. Food product according to claim 1, characterised in that it is one fruit-based.
 17. Food product according to claim 1, characterised in that it is based on several fruits.
 18. Food product according to claim 1, characterised in that the fruit or fruits have a high organic acid content.
 19. Food product according to claim 18, characterised in that the fruits are: orange, lemon, grape, pineapple, apple, pear, peach and/or red berries.
 20. Food product according to claim 1, characterised in that it further contains milk and/or a vegetable juice.
 21. Food product according to claim 20, characterised in that the vegetable juice is a juice made from soybean.
 22. Food product according to claim 1, characterised in that the organic acids are malic acid, citric acid, tartaric acid, pyruvic acid, fumaric acid and/or gluconic acid.
 23. Method of preparing a food product including the following steps: a) Depletion of organic acids from the fruit-based matrix, b) Addition of probiotics to the matrix obtained at step a), c) Packaging of the product obtained after step b).
 24. Method according to claim 23, characterised in that step a) for depleting organic acids from a fruit-based matrix is carried out by selecting a fruit matrix having low natural acidity.
 25. Method according to claim 24, characterised in the selection of a fruit matrix having a low natural acidity is carried out via varietal selection of the fruits and/or by controlling the ripening of the fruit.
 26. Method according to claim 23, characterised in that the step a) for depleting organic acids from a fruit-based matrix is carried out via de-acidification of the fruit matrix.
 27. Method according to claim 26, characterised in that the reduction in titratable acidity of the fruit matrix is carried out via electrodialysis of the fruit matrix, precipitation of the organic acids from the fruit matrix with calcium salts, malolactic fermentation, selective assimilation of the citric acid and/or passing the fruit matrix over an anion exchange resin.
 28. Method according to claim 23, characterised in that step b) and step c) are carried out simultaneously.
 29. Method according to claim 23, characterised in that the fruit-based matrix is a fruit juice, a concentrate-based reconstituted fruit juice, or a fruit puree.
 30. Method according to claim 23, characterised in that step for adding lactic acid is carried out between step b) and step c) of said method, or simultaneously therewith.
 31. Strain of Lactobacillus plantarum deposited at the Collection Nationale des Cultures de Microorganismes on 4 Apr. 2002, under the number CNCM 1-845. 